Genome-wide Association Studies Of Specific Anti-nuclear Autoantibody Sub-phenotypes In Primary Biliary Cholangitis

Primary biliary cholangitis?PBC?is an autoimmune disease characterized by abnormal elevation of serum alkaline phosphatase,destruction of intra-hepatic small-to-medium-sized bile ducts and anti-mitochondrial antibody?AMA?production.In addition to AMA,autoantibodies against sp100 and gp210,two subtypes of antinuclear antibodies?ANA?,are important serological markers of PBC and appear to be nearly 100%specific for PBC.Both sp100 and gp210 are present in approximately 10%to 40%of patients and may correlate with disease prognosis.Genetic studies emphasize strong genetic predisposition in the PBC development among different ethnical groups.At present,the genetic basis of ANA production in PBC is not clear.Identification of genetic factors associated with ANA production will help us to understand the pathogenesis of the disease.The objective of this study is to identify major genetic determinants associated with the production of autoantibodies against sp100 and gp210.Methods are as follows:1.qualitative detection of AMA-M2,sp100 and gp210 autoantibodies in PBC patient serum samples using enzyme linked immunosorbent assay.2.genome-wide single nucleotide polymorphism?SNP?genotyping was performed using HumanOmniZhongHua-8 beadchip from Illumina.PLINK1.07 software was used to perform genome-wide association?GWA?analysis for ANAs?anti-sp100 and anti-gp210 antibodies?in 930 PBC patients after quality control.3.TaqMan genotyping and Sanger sequencing methods were used for SNP validation in a separate PBC cohort consisting 1,252 patients.4.SNP,human leukocyte antigen?HLA?classical alleles and amino acid variants of HLA antigens were imputed using SNP2HLA with a large Han-MHC?major histocompatilibity complex?database as a reference panel.5.association analysis and conditional regression analysis were performed to identify major genetic determinants of anti-sp100 and-gp210 sub-phenotypes in the MHC region.GWA analysis showed significant association of multiple SNPs with the production of anti-sp100,but not anti-gp210 autoantibodies.All SNPs significantly associated with anti-sp100 antibody were located in the MHC region.The strongest association was with rs1794280(P=4.83×10^-10,odds ratio[OR]=2.79,95%confidence interval[CI]=2.00-3.90).Linkage disequilibrium?LD?analysis among most significant SNPs identified two independent LD groups,with rs492899 and rs1794280 as tagSNPs,respectively.In a separated PBC cohort consisting of 234 anti-sp100 positive and 1,018 anti-sp100 negative patients,rs492899(P=6.88×10^-13,OR=2.82,95%CI=2.13-3.75)and rs1794280(P=4.25×10^-21,OR=4.85,95%CI=3.49-6.73)were verified as significantly associated with anti-sp100 antibodies.Meta analysis with 445 anti-sp100 positive and 1,729 anti-sp100negative PBC samples showed combined P value with 3.27×10^-22?OR=2.90,95%CI=2.34-3.66?for rs492899 and 5.78×10^-28?OR=3.89,95%CI=3.05-4.96?for rs1794280.To further map additional SNPs,HLA classical alleles and amino acid variants in HLA antigens,we performed imputation analysis using 7,889 SNPs in MHC region from 922 PBC samples and Han-MHC database.Association analysis with 211 anti-sp100 positive and 711 negative cases found that the most significant HLA allele was DRB1*03:01(P=1.51×10^-9,OR=2.97,95%CI=2.06-4.29),and the most significant amino acid polymorphism was DR?1-Asn77/Arg74,which was completely linked with DRB1*03:01 and had the same signal.Conditional regression analysis showed that alleles DRB1*03:01,DRB1*15:01,DRB1*01and DPB1*03:01 explain almost all associations of HLA with anti-sp100 antibody sub-phenotype.DR?1-Asn77/Arg74,DR?1-ser37 and DP?1-lys65 were the main genetic variants accounting for anti-sp100 antibody sub-phenotype.This study indicated significantly genetic predisposition to sp100 autoantibody,but not gp210 autoantibody,sub-phenotype in PBC patients.HLA classical alleles and amino acid variations associated with anti-sp100 antibody were explained,suggesting the importance of specific HLA antigen variants in the process of anti-sp100 antibody production.The mechanisms of sp100 and gp210 autoantibody production were likely highly different.The striking difference in genetic predisposition observed between sp100 and gp210sub-phenotypes may hint distinct roles of these two autoantibodies in PBC pathogenesis.